eSymposia | Myeloid Cells and Innate Immunity in Solid Tumors

Sep 21, 2020 ‐ Sep 23, 2020



Sessions

Tumor-Immune Communication Via Extracellular Vesicles: A Lymph Node Macrophage Link

Sep 16, 2020 7:00pm ‐ Sep 16, 2020 7:00pm

Speaker(s):

Macrophage proliferation machinery drives immunosuppression and tumor progression

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Macrophage proliferation machinery drives immunosuppression and tumor progression Chong Zuo1, Brett Knolhoff1, John Baer1, Jad Belle1, Christina Fu2, David DeNardo1,3 1Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA 2Grinnell College, Grinnell, IA, 50112,USA 3 Alvin J. Siteman Comprehensive Cancer Center, St. Louis, MO 63110, USA Tumor associated macrophages (TAMs) are involved in many aspects of cancer development and are frequently negatively correlated with cancer patient outcomes. We have previously found that in pancreatic ductal adenocarcinoma (PDAC), a significant percentage of TAMs are highly proliferative. To better understand the impacts of macrophage proliferation on macrophage phenotypes, numbers and PDAC patient outcomes, we used parallel studies in human PDAC tissues and genetically engineered mouse models (GEMM) to model both PDAC and manipulate macrophage proliferation. First, we identified cancer-associated fibroblasts (CAFs), rather than cancer cells, as the primary drivers of macrophage proliferation through the production of macrophage colony-stimulating factor (M-CSF). We also observed that proliferating macrophages in single cell RNA sequencing data (ssRNAseq) from human and mouse tumors have enhanced inflammatory and immunosuppressive phenotypes. To examine whether limiting proliferation of TAMs would alter PDAC progression in vivo, we developed a GEMM to manipulate expression of the cell cycle inhibitor p21. Unexpectedly, expression of p21, while limiting macrophage proliferation, enforced macrophage immunosuppressive capacity and led to loss of T cell tumor control and increased tumor progression. In in vitro models and in vivo ssRNAseq data from mouse PDAC, we observed the expression level of p21 in macrophages is linked to elevated expression of both inflammatory and immunosuppressive cytokines. This observation also holds true in analyzing samples from human PDAC patient, stratification of TAMs by either proliferation or p21 expression identifies a subset of highly inflammatory TAMs. Finally, we have found that treatment with chemotherapy induces p21 expression in macrophages. Taken together, these data suggest that the macrophage proliferation machinery, including p21, is a critical regulator of immune suppression in PDAC tumors and that chemotherapy, while killing tumor cells, may ultimately enforce this myeloid driven immune suppression.

Speaker(s):
  • Chong Zuo, BS, Washington University in Saint Louis

A targetable, myeloid orchestrated immunosuppressive microenvironment characterizes bone metastatic human prostate cancer

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

A targetable, myeloid orchestrated immunosuppressive microenvironment characterizes bone metastatic human prostate cancer Ninib Baryawno 1,2,3,4,*, Youmna Kfoury 1,2,3,*, Nicolas Severe 1,2,3,*, Shenglin Mei 5,*, Karin Gustafsson 1,2,3,*, Taghreed Hirz 1,2,3,*, Thomas Brouse 1, Elizabeth W. Scadden 1, Anna A. Igolkina 6, Bryan D. Choi 7, Nikolas Barkas 5, John H. Shin 7,†, Philip J. Saylor 8,†, David T. Scadden 1,2,3,†, David B. Sykes 1,2,3,†, Peter V. Kharchenko 2,5,†, as part of the Boston Bone Metastasis Consortium 1 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA 2 Harvard Stem Cell Institute, Cambridge, MA, USA
 3 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 4 Childhood Cancer Research unit, Dep. Of Children’s and Women’s Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden 5 Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA 6 St. Petersburg Polytechnical University, St. Petersburg, Russia 7 Department of Neurosurgery, Harvard Medical School, Boston, MA, USA 8 Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, USA *,† Contributed equally Bone is the third most common site for solid tumor metastases with the highest incidence for prostate and breast cancer. Current treatments are rarely curative (Migliorini et al., 2020) and patients are at a disadvantage when it comes to response to immune check point therapies (Beer et al., 2017). It is not clear what elements of the complex bone microenvironment contribute to it being a favored and treatment refractory metastatic site. We conducted a high-resolution analysis, using droplet-based single cell RNA sequencing of patient bone metastatic prostate cancer samples (Tumor=9) paired with bone marrow (BM) from the same patient that was adjacent (Involved=8) or distant (Distal=8) to the site of metastases. These were contrasted with BM samples from patients undergoing hip replacement surgery representing a non-malignant, inflammatory microenvironment (Benign=7) in addition to a published data set of healthy BM. This multi-layered approach allowed us to control for inter-patient variation and identify cellular and molecular changes that are specific to a malignant metastatic microenvironment rather than a general reflection of an inflammatory state. Analyses included hematopoietic lymphoid and myeloid cells, in addition to non-hematopoietic clusters representing tumor cells, mesenchymal and endothelial lineages. The presence of metastases reshaped the immune cell composition of the BM when compared to benign samples as reflected by a striking depletion of B cells and B cell progenitors in all three patient derived fractions, a substantial population shift within the myeloid compartment of the tumor fraction and an expansion of dysfunctional T cell clusters characterized by decreased cytotoxicity and increased exhaustion signatures. In particular, tumor associated M2 polarized macrophages (TAM) and tumor inflammatory monocytes (TIM) were selectively enriched in the tumor fraction. Their gene expression characteristics suggested an immune suppressive function. Indeed, the increased proportion of TAMs and TIMs correlated with cytotoxic T cell exhaustion and a reduced cytotoxicity signature respectively. Ligand/ receptor interaction analysis identified 241 potential channels of interaction between the myeloid and lymphoid compartments. To validate our findings, we developed a C57BL/6 syngeneic mouse model of highly penetrant bone metastatic prostate cancer that produces bone osteolytic and osteoblastic lesions. Using this model, we demonstrated that disrupting one of the channels mediating myeloid/lymphoid interaction by either blocking the ligand or deleting the receptor, the survival of bone metastases bearing mice improved. Further, T regulatory cells decreased as did T cell exhaustion as demonstrated by single cell RNA sequencing, phenotypic and functional assays. These data provide a high resolution landscape of the bone metastatic prostate cancer microenvironment that is distinct from benign inflammation and demonstrate the existence of a targetable immunosuppressive milieu that may guide novel therapeutic strategies to this devastating medical condition.

Speaker(s):

Macrophage-associated genes to predict chemotherapy resistance in breast cancer patients

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Macrophage-associated genes to predict chemotherapy resistance in breast cancer patients Cigdem Selli1, Bin-Zhi Qian1 1 MRC Centre for Reproductive Health, College of Medicine and Veterinary Medicine, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK Rational: Gene expression profiles of breast tumours under neoadjuvant chemotherapy have been recently identified (NEO study) 1. In NEO study, those patients achieved a complete response were classified as “responder” whereas those with progressive disease were classified as “non-responder”. Tumours were sequentially biopsied and profiled at pre-treatment, early on treatment, mid-chemotherapy, and at surgical resection. It is well established that tumour-associated macrophages promotes chemotherapy resistance in preclinical studies 2. We propose that macrophage-associated genes can be used to pinpoint the lack of response to chemotherapy after a relatively short duration of treatment (2 weeks or mid-chemotherapy) contributing to the individualized treatment of patients and improving patient outcomes. Approach: To delineate macrophage-associated genes in chemotherapy resistance, we determined the macrophage-correlated genes specific to non-responder tumours in NEO study using R and Bioconductor. Macrophage abundance was estimated using ImSig, a computational approach to estimate immune cell abundance from gene expression 3. Mean expression of macrophage genes of ImSig and Pearson correlation were used to identify macrophage-associated genes in each group. Results: Macrophage abundance was similar across groups in all timepoints suggesting presence of a subset of tumour-associated macrophages rather than overabundance of macrophages drives therapy resistance. Based on this, top macrophage-correlated genes in each group were determined. Then, non-responder-specific genes were identified to determine resistance-driving macrophage-associated genes. Among these genes, those with significantly higher levels in non-responders compared to responders were determined as candidates. In addition, candidate genes were confirmed to be macrophage-specific by comparing with Human Protein Atlas data and literature search. Future perspectives: Candidate genes will be validated using gene expression data from I-SPY 1 trial 4 with a total of 248 sequential samples from responder and non-responder patients. Candidate genes will be further validated at protein level by staining using NEO study samples. This will allow identification of robust macrophage-associated genes allowing early prediction of lack of response to chemotherapy contributing to the timely detection of resistance development and better individualized treatment of patients. References 1. Bownes RJ et al 2019 Breast Cancer Res 21(1) 2. Ali HR et al 2016 Plos Med 13(12) 3. Nirmal AJ et al 2018 Cancer Immunol Res 6(11) 4. Magbanua MJ et al 2015 Breast Cancer Res 17

Speaker(s):

Macrophage-dependent regulation of breast cancer iron homeostasis

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Macrophage-dependent regulation of breast cancer iron homeostasis Daniel Greiner and Minna Roh-Johnson Triple negative breast cancer (TNBC) cells have high metabolic requirements, particularly for metabolites that drive redox reactions such as iron. Iron is so critical to cellular growth that sequestering iron away from a tumor can reduce numerous tumor proliferation in vitro. Despite these findings, pharmacological efforts to chelate iron and inhibit tumor progression in vivo have remained unsuccessful. To improve these efforts, we seek to understand how the microenvironment affects tumor iron homeostasis. Tumors acquire the majority of their iron from macrophages in the tumor microenvironment; Macrophages store high amounts of iron and can secrete iron in the presence of a tumor. In addition to iron secretion, macrophages can also promote tumor growth and cancer progression through both secreted signals and contact-mediated mechanisms. We will determine whether macrophage-tumor paracrine signaling can disrupt tumor iron homeostasis, affecting metastasis. Furthermore, we have identified that macrophages can transfer cytoplasm to cancer cells during metastasis. Thus, we will test whether macrophages make iron available to tumor cells through contact-mediated mechanisms, independent of secreted iron. To address these questions, we use primary human blood monocyte derived macrophages and TNBC cells in coculture to identify how changes occur in tumor iron homeostasis. Our preliminary data suggest macrophages may cause changes to the amount of available iron to the tumor, driving changes to tumor iron import and export. Furthermore, our data reveal a novel form of regulation in which macrophages may disrupt tumor iron homeostasis through direct transfer of iron or iron-associated proteins. Taken together, our findings suggest that macrophages promote iron dysregulation during tumor progression. Our future efforts will focus on determining the signaling mechanisms behind these changes, in an effort to identify potential drug targets that will prevent macrophage-dependent changes to tumor iron homeostasis.

Speaker(s):

The role of ZNRF3 and TP53 inactivation in adrenocortical tumorigenesis

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

The role of ZNRF3 and TP53 inactivation in adrenocortical tumorigenesis Authors list: 1- James Wilmouth Jr, 1- Julie Olabe, 2- Kaitlin J. Basham, 1- Laly Pucheu, 1- Florence Roucher-Boulez, 1- Cecily Lucas-Rodrigues, 1- Antoine Martinez, 2- Gary Hammer, & 1- Pierre Val Affiliations: 1- Génétique Reproduction et Développement, UMR 6293, Clermont-Ferrand Cedex, France 63000, 2- Endocrine Oncology Program, University of Michigan Rogel Cancer Center, Ann Arbor, MI 48109. Abstract: Adrenocortical carcinoma (ACC) is an infrequent and aggressive cancer that originates from steroidogenic cells within the adrenal cortex. Half of patients present with metastatic spread at initial diagnosis, resulting in a low 5-year survival rate. Recent genomic analysis identified the most common alteration in ACC as inactivation of the transmembrane E3 ubiquitin-ligase Zinc and Ring Finger 3 (Znrf3), a potent tumor suppressor responsible for regulating the WNT/beta-catenin pathway. In order to study the role of Znrf3 inactivation in the adrenal cortex, we generated a steroidogenic factor-1 (Sf-1) Cre mediated Znrf3 knockout mouse model. The resulting mice developed hyperplasia associated with moderate increase in WNT/beta-catenin signaling by 6 weeks; however, this was followed by regression characterized by the induction of senescence and immune recruitment. In an attempt to surpass this senescent phenotype, we developed a mouse model derived from the most aggressive subgroup of ACC, composed of p53 & Znrf3 inactivation. TP53 is involved in p21-induced senescence, therefore we hypothesize that p53 ablation in the context of Znrf3 inactivation will surpass the senescent phenotype and allow tumorigenesis to proceed. We show that inactivation of p53 & Znrf3 in the mouse adrenal cortex does not completely surpass the immune recruitment and senescent phenotype caused by Znrf3 inactivation alone; however, these double KOs eventually form aggressive carcinomas that metastasize to the lungs, liver, and peritoneal. Interestingly, these aggressive tumors are associated with immune cell exclusion at primary and potentially secondary locations. Our current aim is to use this ACC mouse model to further investigate whether immune exclusion is required for tumor progression, and whether steroid secretion plays a role in this process.

Speaker(s):

Engineering Nanoshapes to Optimize Tumor Associated Macrophage Re-Education

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Engineering Nanoshapes to Optimize Tumor Associated Macrophage Re-Education Jessica A. Widman and Laura J. Suggs Department of Biomedical Engineering, The University of Texas at Austin Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that is unresponsive to most targeted treatment options, including current immunotherapies. Additionally, TNBC survival rates are inversely correlated with the immunosuppressive tumor associated macrophage (TAM) population. Thus, an emerging immunotherapeutic strategy is re-educating the TAM population towards an inflammatory, anti-tumor phenotype. Due to the phagocytic nature of TAMs, and the enhanced permeability and retention effect, nanocarriers have been used to deliver TAM-re-educating therapeutics. However, the efficacy of these nanocarriers are limited by their tumor accumulation, tumor penetration, and TAM uptake rate. We are engineering three distinctly shaped nanoparticles to investigate how nanoparticle shape influences tumor accumulation, tumor penetration, and TAM uptake rate to optimally deliver TAM-targeted therapeutics in a TNBC model. We hypothesize that the short dimensions of nanorods and nanotriangles will promote tumor penetration while the long dimensions will improve circulation time and tumor accumulation. We have synthesized nano-rods, -triangles, and -shells, which have one, two and three long dimensions, respectively. They have equivalent long dimension lengths of approximately 75 nm. We have shown that our nanoshapes are cytocompatible and can be imaged in cells without the addition of an exogenous fluorescent molecule. To our knowledge, these nanoshapes are the first otherwise physicochemically equivalent nanoparticles synthesized to study the fundamental impact of shape on TAM nanoparticle uptake and distribution. These nanoshapes are size tunable; inherently excited by 2-photon imaging; cytocompatible; and capable of carrying a drug molecule or targeting ligand, such as CpG. Future studies include investigating the impact of nanoparticle shape on macrophage uptake rate in vitro and investigating spatial and cellular distribution in a TNBC model in vivo. Additionally, we will explore the use of laser irradiation and CpG conjugation with these nanoshapes to induce an effective immune response in a TNBC model.

Speaker(s):

PKC Delta Activation Restricts Innate Immune Suppression and Promotes antigen Presentation in Triple Negative Breast Cancer

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

PKC Delta Activation Restricts Innate Immune Suppression and Promotes antigen Presentation in Triple Negative Breast Cancer Immune checkpoint blockade (ICB) has revolutionized cancer therapy showing unprecedented long-term antitumor responses. However, most patients do not respond to ICB therapies due at least partly to immunosuppression. Immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses both innate and adaptive immunity. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to ICB. On the other hand, cross-presenting dendritic cells (DCs) are essential to generate an antitumor immune response. Breast cancer patients who harbor higher numbers of these DCs (cDC1) have a better prognosis than patients with lower DC numbers. Several strategies aiming at achieving an effective combination with immunotherapy are under active investigation. The central dogma of these strategies consists of inducing T cells into “immunologically cold tumors” which are defined by having low neoantigen burden and a paucity of T cells and DCs. Hence, strategies that enhance cross-presenting DCs and T cell antitumor potential while altering MDSC’s suppressive function are likely to be effectively combined with immunotherapy for a maximum therapeutic benefit. Protein Kinase C (PKC) is a family of kinases composed of 11 isoforms that play a critical role in cell signaling. PKC delta (PKCd) is the most abundant isoform in myeloid cells and plays an important role in dendritic cell (DC) function. To date, the role of PKCd in myeloid cells in cancer is unknown. Using varied informatic approaches in patient databases, we found that BC patients with both high expression of PRKCD (PKCd gene) and either high expression of CD8+ T cell or low expression of MDSC gene signatures in tumors had a significantly greater overall survival compared to other groups, suggesting support for activation of PKCd. Novel preliminary data suggests that PKC agonism using FDA-approved PEP005 and prostratin reduced MDSC generation from BM progenitors specifically via activation of PKCd isoform. PKC agonism induced MDSC differentiation to Ly6C+ CD103+ DCs both ex-vivo and in adoptive transfer experiment. Additionally, PEP005-treated MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. Treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumors. These findings propose PKCd as a novel target in myeloid cells to tip the balance from immune suppression to effective antitumor immunity.

Speaker(s):
  • Mehdi Chaib, M.S., University of Tennessee Health Science Center

B Cell-Like Neutrophil Found in Peripheral Blood of Melanoma Patients

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

B Cell-Like Neutrophil Found in Peripheral Blood of Melanoma Patients Melissa A. Meyer1*, Huy Q. Dinh1,2*, Yanfang Zhu1,3, Shu Liang1, Gregory Seumois1, Pandurangan Vijayanand1, Sergio Catz4, Christian Ottensmeier5, and Catherine C. Hedrick1 1Center for Cancer Immunotherapy, La Jolla Institute for Immunology, La Jolla, CA, USA, 2McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA, 3Department of Pediatrics, University of California San Diego, La Jolla, CA, USA, 4Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA, 5University of Southampton, Southampton, UK. *Equal contributions Neutrophils engage both pro- and anti-tumoral functions. We hypothesized that the heterogeneity observed amongst neutrophils in the periphery during cancer is seeded by heterogeneity amongst immature neutrophils populations in the bone marrow (BM). To test this, we employed single-cell transcriptomics to profile immature neutrophils from the healthy human BM. We identified a novel neutrophil subset that expresses several B cell markers, including CD79A/B, components of the B cell receptor and PAX5, a B cell-specific transcription factor. We termed this population BNeuts. BNeuts are more like neutrophils than B cells, with a neutrophil-like transcriptome, a large, granular cytoplasm, and a superior ability to engage NETosis. Using lineage-tracing studies, we determined that BNeuts derive from early human neutrophil progenitors rather than human B cell progenitors. We found that BNeuts are confined to the BM of healthy individuals but expand into the blood of melanoma patients, specifically in early-stage disease. BNeuts are also found in the tumors of head and neck cancer patients, showing they can infiltrate tumor tissue. To understand why neutrophils might employ a B cell program, we tested classical B cell functions within this cell type. We found BNeuts engaged an antigen presentation program, a function that is usually absent in neutrophils. We show that BNeuts phagocytize tumor cells, have increased expression of antigen presentation-related genes compared to other neutrophils, and modulate CD4+ T cell responses. Together, we have identified a novel neutrophil subset expressing B cell markers that is preferentially expanded in the blood of melanoma patients and poised to control T cell responses in cancer.

Speaker(s):

Interferon epsilon alters peritoneal myeloid cell populations in a murine model of ovarian cancer

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Interferon epsilon alters peritoneal myeloid cell populations in a murine model of ovarian cancer Nicole K. Campbell, Zoe R.C. Marks & Paul J. Hertzog Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia. The novel type I interferon, interferon epsilon (IFNε), is a unique cytokine which is constitutively expressed by epithelial cells under hormonal regulation in the female reproductive tract (FRT)1. Although IFNε is known to be protective against FRT infections 2, its role in the anti-cancer immune response remains unknown. Ovarian cancer is a common yet lethal FRT cancer, which metastasises to the peritoneal cavity in the majority of cases. Preliminary research has suggested that IFNε may protect against metastasis of ovarian cancer through action on both tumour and immune cells. Here, we have investigated the activity of IFNε in an orthotopic mouse model of ovarian cancer, using the ID8 tumour cell model. IFNε treatment was found to significantly reduce tumour burden, ascites development and peritoneal hemorrhaging in challenged mice, compared to both IFNβ and vehicle control (PBS). This was accompanied by stark differences in peritoneal immune cell populations; IFNε treatment increased numbers of resident large peritoneal macrophages, and reduced infiltration of pro-inflammatory small peritoneal macrophages. Furthermore, while a population of myeloid derived suppressor cells (MDSC) was observed in tumour-bearing mice, these MDSC were absent in mice which received IFNε treatment. These results indicate that modulation of myeloid cell populations in the peritoneal cavity may contribute to the efficacy of IFNε in preventing peritoneal metastasis of ovarian cancer. Further investigation into the immunomodulatory activity of IFNε within the peritoneal cavity is required to elucidate the potential of IFNε to be used to treat ovarian cancer and other peritoneal pathologies. 1. Marks, Z. R. C. et al. PROPERTIES AND FUNCTIONS OF THE NOVEL TYPE I INTERFERON EPSILON. Semin. Immunol. 43, 101328 (2019). 2. Fung, K. Y. et al. Interferon-ε protects the female reproductive tract from viral and bacterial infection. Science (80-. ). 339, 1088–1092 (2013).

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